This application is the national phase under 35 U.S.C. xc2xa7371 of PCT International Application No. PCT/JP97/04111 which has an International filing date of Nov. 12, 1997, which designated the United States of America.
The present invention relates to a novel Semaphorin belonging to the Semaphorin family and a gene therefor. More particularly, it relates to a novel Semaphorin having neurite outgrowth inhibition activity and proteins analogous thereto, or peptide fragments of, or antibodies against, such proteins, genes (DNAs) encoding such proteins, expression vectors for said genes, transformed cells into which said expression vectors have been introduced, methods for producing a recombinant protein which employ said transformed cells, antisense nucleotides against the above genes, transgenic animals involving insertion or deletion of the above genes, or screening methods for antagonists of the above proteins, and it further relates to use of such proteins, peptides, antibodies, genes, antisense nucleotides or the like as pharmaceutical or diagnostic agents or laboratory reagents.
In 1992, Fasciclin IV (latterly called G-Sema I) was cloned as one of the genes involved in guidance for neuron in grasshopper. The next year, the existence of a gene family of which members encode analogous domains and are distributed in a wide range of species covering insects, viruses, nematodes, and human was revealed, and the gene family members were designated xe2x80x9cSemaphorin genesxe2x80x9d. To date, more than ten genes belonging to the Semaphorin family have been reported (Cell, 81, 471-474 (1995)).
These Semaphorin genes characteristically contains, in the amino acid sequences which they encode, similar structures called semaphorin domain each consisting of about 500 amino acids (Neuron, 14, 941-948 (1995); Cell, 75, 1389-1399 (1993)). Although the homologies of the above amino acid sequences among Semaphorins are 80-20% and are thus not always high, some of the amino acid residues are extremely well conserved as exemplified by thirteen cysteine residues located at conserved positions. In the regions other than semaphorin domains, Semaphorins are highly varied one another. Specifically, Semaphorins include both of secretory and membrane-bound forms, and have various structures including those having Ig domains, thrombospondin domains, or a cluster of basic amino acids at its carboxy terminus.
Among such Semaphorins, functions have been verified for only a few, including, for example, Fasciclin IV of grasshopper, Semaphorins I and II of drosophila, Collapsin of chick, and Semaphorin III which corresponds to Collapsin in mammals. All of these Semaphorins are, however, known to inhibit axon outgrowth and synapsis formation during the stage of ontogenesis, that is, in the course of the neural network formation at the embryonic or fetal stage (Neuron, 14, 941-948 (1995); Neuron, 14, 949-959 (1995); Cell, 81, 631-639 (1995); Cell, 75, 1389-1399 (1993); Cell, 75, 217-227 (1993); and Neuron, 9, 831-845 (1992))
Although Semaphorin genes are known to perform its function at the ontogenetic stage as described above, it has not yet been ascertained whether or not they perform any function also in the adult. However, some Semaphorin genes are known to be expressed also in the adult in which formation of the neural network has been already completed, suggesting that they may have some function also in said adult. For example, the central nervous system (CNS) in the adult is widely known to lack regenerating ability, and some Semaphorins which inhibit neurite outgrowth may conceivably function as a CNS-neuron regeneration inhibitor in the adult (Nature, 378, 439-440 (1995)). In addition, it has been suggested, by a recently reported study on Semaphorin III-knockout mouse, that a certain Semaphorin may probably act in inhibiting the growth of cardiac muscles (Nature, 383, 525-528 (1996)). Furthermore, a certain Semaphorin has been suggested to be involved in survival and aggregation of B lymphocytes (Proc. Natl. Acad. Sci. USA, 93, 11780-11785 (1996)).
It is thus being demonstrated that Semaphorins play important roles not only in the nervous system but also in non-nervous systems, and therefor attracting great interest in studies on said Semaphorins.
Accordingly, an object of the present invention is to provide a novel Semaphorin and proteins analogous thereto, peptide fragments of, or antibodies against, such proteins, genes (DNAs) encoding such proteins, expression vectors for said genes, transformed cells into which said expression vectors have been introduced, methods for producing a recombinant protein which employ said transformed cells, antisense nucleotides against the above genes, transgenic animals involving insertion or deletion of the above genes, or screening methods for antagonists of the above proteins, all of which are useful for medical treatments, diagnoses, or studies of neurological diseases, and to further provide use of such proteins, peptides, antibodies, genes, antisense nucleotide or the like as pharmaceutical or diagnostic agents or laboratory reagents.
Despite the increasing interest in studies on Semaphorin as described above, not all structures of the presumably more than twenty kinds of Semaphorin genes have been elucidated. The present inventors have planed to clone unknown Semaphorins by making use of gene regions homologous among known Semaphorins. Firstly, we aimed at a region homologous between Collapsin derived from chick and G-Sema I derived from grasshopper, and used it to prepare synthetic primers for amplifying a fragment corresponding to this region. Although it was uncertain whether or not the use of these synthetic primers may result in successful cloning of any novel Semaphorin other than Collapsin and G-Sema I, we performed PCR using the synthetic primers with cDNAs derived from mouse embryo as a template. As a result, we have succeeded in cloning a novel Semaphorin gene.
Analysis revealed that the novel Semaphorin of the present invention contains no transmembrane regions in its structure, indicating that it belongs to the secretory Semaphorin subfamily. Although five to six Semaphorins of the secretory type have been hitherto known, only one of such Semaphorins has demonstrated activities. The Semaphorin of the present invention is of the secretory type, and therefore, is characterized in that it may serve, as such, as a pharmaceutical or diagnostic agent or laboratory reagent in the art.
It was also shown, by further investigations on the Semaphorin of the present invention, that it inhibits neurite outgrowth, and that expression of the gene begins at the embryonal stage, and in the adult, the gene is characteristically expressed in a wide range of central and peripheral tissues in a localized manner.
Thus, the gist of the present invention relates to:
(1) a gene encoding the following protein (a) or (b):
(a) a protein comprising the amino acid sequence shown in SEQ ID NO: 1,
(b) a protein which comprises an amino acid sequence wherein one or more amino acids are deleted, substituted and/or added in the amino acid sequence shown in SEQ ID NO: 1, and which protein has neurite-outgrowth inhibition activity;
(2) a gene comprising the following DNA (a) or (b):
(a) DNA comprising the base sequence shown in SEQ ID NO: 2,
(b) DNA which hybridizes under stringent conditions to DNA comprising the base sequence shown in SEQ ID NO: 2, and which encodes a protein having neurite-outgrowth inhibition activity;
(3) DNA which is cloned from a human cDNA library or a human genomic library, and which hybridizes under stringent conditions to DNA comprising at least part of DNA consisting of the base sequence shown in SEQ ID NO: 2;
(4) a protein obtained by expressing either a gene of the above item (1) or (2), or DNA of the above item (3);
(5) an expression vector which contains either a gene of the above item (1) or (2), or DNA of the above item (3);
(6) a transformant obtained by introduction of an expression vector of the above item (5);
(7) a transformant of the above item (6) in which a gene of the above item (1) or (2), or DNA of the above item (3), existing in an expression vector of the above item (5) is stably retained;
(8) a process for producing a recombinant protein, which process comprises culturing a transformant of the above item (6) or (7), and recovering the recombinant protein expressed;
(9) a peptide comprising a segment of at least six or more amino acids of a protein of the above item (4);
(10) an antisense nucleotide, or chemically modified variant thereof, which is directed against a segment of at least eight or more bases of a gene of the above item (1) or (2), or of DNA of the above item (3);
(11) an antisense nucleotide, or chemically modified variant thereof, of the above item (10), which inhibits expression of a protein of the above item (4) when introduced into cells;
(12) an antibody or its fragment which specifically binds to a protein of the above item (4) or to a peptide of the above item (9);
(13) a pharmaceutical agent comprising, as an active ingredient, a gene of the above item (1) or (2), DNA of the above item (3), a protein of the above item (4), a peptide of the above item (9), an antisense nucleotide or chemically modified variant thereof of the above item (10) or (11), or an antibody or its fragment of the above item (12);
(14) a neurite outgrowth inhibitor for PNS-neurons, characterized in that it contains at least one of the proteins of the above item (4);
(15) a screening method for antagonists of a protein comprising the amino acid sequence shown in SEQ ID NO: 1, characterized in that it employs a protein of the above item (4); and
(16) a transgenic animal in which either a gene of the above item (1) or (2), or DNA of the above item (3) has been artificially inserted into its chromosome, or has been knocked out.
For the purpose of the present invention, xe2x80x9cgenexe2x80x9d is also referred to as xe2x80x9cDNAxe2x80x9d. The gene is not specifically restricted so far as it encodes a protein having the neurite-outgrowth inhibition activity according to the novel Semaphorin. Examples are novel mouse Semaphorin genes such as a gene encoding a protein which comprises the amino acid sequence shown in SEQ ID NO: 1 of the sequence listing or a gene comprising the base sequence shown in SEQ ID NO: 2 of the sequence listing. Also included in the genes of the present invention are those genes encoding a protein which comprises an amino acid sequence wherein one or more amino acids are deleted, substituted and/or added in the amino acid sequence shown in SEQ ID NO: 1, and those genes which hybridize under stringent conditions to DNA comprising the base sequence shown in SEQ ID NO: 2, provided that they encode proteins having neurite-outgrowth inhibition activity. These genes are explained below in order.
1) Gene Encoding A Novel Mouse Semaphorin
Of the above-mentioned genes, xe2x80x9ca gene which encodes a protein comprising the amino acid sequence shown in SEQ ID NO: 1xe2x80x9d or xe2x80x9ca gene comprising the base sequence shown in SEQ ID NO: 2xe2x80x9d is a novel mouse Semaphorin gene cloned in the present invention.
Such genes may be cloned, for example, as follows: PCR may be performed using single-stranded cDNAs derived from mouse embryo as a template with oligonucleotide primers which can amplify the region homologous between two known Semaphorins, in this case Collapsin and G-Sema I, and the resulting amplified fragment may be used as a probe for screening cDNA libraries such as those derived from adult mouse brain to clone the desired gene. Particular techniques for such cloning may be found in the standard texts such as xe2x80x9cMolecular Cloningxe2x80x9d, 2nd ed., Cold Spring Harbor Laboratory Press (1989). PCR may be performed, for example, by making reference to xe2x80x9cPCRxe2x80x9d, IRL Press (1991). The base sequence of cloned DNA may be determined by conventional methods, for example, using a sequencing kit commercially available.
When compared with previously reported Semaphorin genes, the novel Semaphorin gene of the present invention exhibits 54% overall identity with mouse Semaphorin D (a mouse homolog of chick Collapsin) at the amino acid level, and exhibits 85% identity with chick Collapsin-5, of which sequence is known only partially, in their semaphorin domains.
2) Gene Encoding Modified Protein of The Novel Semaphorin
Of the above-mentioned genes, xe2x80x9ca gene encoding a protein which comprises an amino acid sequence wherein one or more amino acids are deleted, substituted and/or added in the amino acid sequence shown in SEQ ID NO: 1 and which protein has neurite-outgrowth inhibition activityxe2x80x9d refers to a gene encoding a so-called xe2x80x9cmodified proteinxe2x80x9d of the novel Semaphorin of the present invention which has neurite-outgrowth inhibition activity. Those skilled in the art may easily obtain a gene encoding such protein, for example, by site-directed mutagenesis (Methods in Enzymology, 100, 448- (1983)) or PCR method (xe2x80x9cMolecular Cloningxe2x80x9d, 2nd ed., Chapter 15, Cold Spring Harbor Laboratory Press (1989); xe2x80x9cPCR A Practical Approachxe2x80x9d, IRL Press, 200-210 (1991)). In this context, the number of amino acid residues to be deleted, substituted and/or added is to be such a number that permits the deletion, substitution and/or addition by a well-known method such as site-directed mutagenesis described above.
For the purpose of the present invention, a protein xe2x80x9cwhich has neurite-outgrowth inhibition activityxe2x80x9d refers to such a protein that has collapse activity on growth cone of neuron or that has neurite-outgrowth inhibition activity. Specifically, these activities may be measured by the following procedures.
The collapse activity on growth cone of neuron may be measured by making reference to M. Igarashi et al., Science, vol. 259, pp. 77-79 (1993)), while the neurite-outgrowth inhibition activity may be measured by making reference to, for example, J. A. Davies et al., Neuron, vol. 2, pp. 11-20 (1990) or M. Bastmeyer, J. Neurosci., vol. 11, pp. 626-640 (1991)).
Briefly, the former activity is measured by adding an expression product derived from the gene of the present invention to neurons cultured under conventional conditions (e.g., xe2x80x9cCulturing, Nerve Cellsxe2x80x9d edited by Banker et al., MIT Press (1991)) in a culture container coated with a substance promoting the neurite outgrowth and the growth-cone formation such as laminin, collagen, polylysine or polyornithine. After the addition, when a sufficient time has passed to induce collapse of growth cone (typically from 30 minutes to one hour after the addition), those neurons are fixed with 1% glutaraldehyde or the like, and the number of the growth cones which have been collapsed is counted under a microscope. Normalization of the samples is typically carried out on the basis of the total amounts of protein included within the samples.
On the other hand, to measure the latter activity, for example, a mass of cells expressing a gene of the present invention described above is co-cultured with neurons in a collagen matrix, and inability of the neurons to outgrow towards said mass of cells expressing the gene or inhibition of the neurite outgrowth is used as an indicator.
Alternatively, a nervous tissue may be trypsinized, and the single cells thus obtained are treated with culture supernatant from cells expressing a gene of the present invention. Significant decrease in the number of neurons having outgrown neurites relative to the control may be used as an indicator.
Examples of modified proteins obtained above are the human and mouse types of modified protein of Semaphorin of the present invention.
3) Gene Hybridizing Under Stringent Conditions To The Novel Semaphorin Gene
Of the above-mentioned genes, xe2x80x9ca gene comprising DNA which hybridizes under stringent conditions to DNA comprising the base sequence shown in SEQ ID NO: 2 and which encodes a protein having neurite-outgrowth inhibition activityxe2x80x9d may refer to any mammal type of the novel Semaphorin such as the human or rat type of Semaphorin of the present invention. In addition, any genes which hybridize under stringent conditions to DNA comprising the base sequence shown in SEQ ID NO: 2 are included in the genes of the present invention, provided that they have neurite-outgrowth inhibition activity. Based on the results of a search through EST database, the human type of novel Semaphorin of the present invention obtained by cloning techniques is believed to comprise, as a partial sequence, the sequence from position 1 to position 200 of Accession Number W16752 or to comprise a sequence extremely analogous (more than 95%, and preferably more than 98% identical) to said sequence. Accordingly, the human type of novel Semaphorin gene of the present invention can be easily cloned on the basis of the EST sequence described above. Libraries used for such screening may be human genomic libraries or human cDNA libraries, and cDNA libraries derived from tissues of human nervous system are preferably used.
As used herein, a gene xe2x80x9cwhich hybridizes under stringent conditionsxe2x80x9d refers to such a gene that hybridizes under conditions described below in Example 1 wherein the salt concentration is 5xc3x97SSPE and the temperature is around 42xc2x0 C.
Also included within the scope of DNA of the present invention are those DNAs cloned from a human cDNA library or a human genomic library which hybridize under stringent conditions to DNA comprising at least part of DNA consisting of the base sequence shown in SEQ ID NO: 2.
Method for cloning such genes may be found, for example, in xe2x80x9cMolecular Cloningxe2x80x9d, 2nd ed., Cold Spring Harbor Laboratory Press (1989), or xe2x80x9cPCRxe2x80x9d edited by McPherson et al., IRL Press (1991). A preferred library used herein is a human genomic library. DNAs of the present invention may also be cloned based on the sequence from position 1 to position 200 of Accession Number W16572 of the EST database, as described above.
The DNAs cloned as above include full-length DNAs. DNA fragments consisting of about 100 bases or more, and a single-stranded forms (coding strands or complementary stands thereof) of said DNA fragments. In some preferred embodiments, they may be genomic DNA such as 5xe2x80x2 transcriptional control region, 3xe2x80x2 transcriptional control region, noncoding sequence of exons, introns or the like. Such sequences which don""t encode any amino acids are also quite useful, for example, in developing a medicine using antisense nucleotides described below.
In the present invention, xe2x80x9cproteinsxe2x80x9d refers to those proteins which may be obtained by expressing the genes (DNAs) of the present invention. A specific example is a novel mouse Semaphorin having the amino acid sequence shown in SEQ ID NO: 1 which is encoded by the longest open reading frame having the base sequence from position 370 to position 2694 of SEQ ID NO: 2. This Semaphorin has a semaphorin domain which corresponds to the amino acid sequence from position 49 to position 572 of SEQ ID NO: 1. The above novel mouse Semaphorin also contains a signal sequence at its N-terminus, and said signal sequence may undergo processing to be removed during its transfer to membrane, resulting in a mature protein. In the case of SEQ ID NO: 1, the mature form is presumed to be a protein consisting of the amino acid sequence beginning at position 20. Since such mature form or modified protein thereof may also be obtained by expressing a gene of the present invention described above, it is also included within the proteins of the present invention.
Expression vectors used in the present invention are not specifically restricted so far as they are expressible vectors into which a gene or DNA of the present invention is inserted, and specific examples are known expression vectors such as pET, pCDM8 and the like.
A transformant of the present invention is obtained by introducing an expression vector described above into desired host cells. Host cells may be prokaryotic or eukaryotic, and are selected depending on the expression vector used. Those transformants in which the foreign gene existing in the expression vector is stably retained in the cells are more preferred.
The expression vector may be suitably introduced into cells by any one of known methods such as the calcium phosphate method, DEAE-dextran method, or electroporation (xe2x80x9cCurrent Protocols in Molecular Biologyxe2x80x9d, F. M. Ausubel et al. ed., John Wiley and Sons (1987)).
A recombinant protein of the present invention is a protein which is obtained by culturing the above transformants and recovering the recombinant protein expressed and which has neurite-outgrowth inhibition activity. Since Semaphorin of the present invention is presumed from its structure to be a secretory protein, the culture supernatant from transformed cells may probably contain the Semaphorin. Therefore, activity measurement of said Semaphorin can be easily carried out as described above by using the culture supernatant as such.
In addition, the recombinant protein produced may be easily purified by a method such as conventional column chromatography or affinity purification using an antibody of the present invention described below.
xe2x80x9cPeptidexe2x80x9d, as used herein, refers to a peptide fragment comprising a segment of at least six amino acids in the amino acid sequence of a protein of the present invention. In this context, the limitation xe2x80x9cat least six amino acidsxe2x80x9d is based on the fact that a minimal size of peptide capable of forming a stable structure consists of six amino acids, and preferred peptides are those consisting of eight or more amino acids, more preferably of about 10-20 amino acids. A short peptide such as those consisting of about 10-20 amino acids can be synthesized on a peptide synthesizer, while a longer peptide can be obtained by preparing DNA through conventional genetic engineering (for example, using treatments with restriction enzymes), and expressing it in, for example, animal cells. The peptide thus prepared may also be modified by conventional methods.
These peptides can be used as pharmaceutical agents as described below, and can also be used for producing antibodies.
xe2x80x9cAntisense nucleotidexe2x80x9d which is directed against a segment of at least eight or more bases in a gene or DNA of the present invention refers to a so-called antisense oligonucleotide, antisense RNA, or antisense DNA, and it may be artificially prepared using a synthesizer, or may be obtained by, for example, expressing a gene in the direction opposite to the usual case (i.e., in the antisense direction). Antisense nucleotides are used for inhibiting expression of Semaphorin of the present invention, and can also be used as laboratory reagents for, for instance, in situ hybridization.
A xe2x80x9cchemically modified variantxe2x80x9d of the above antisense nucleotide refers to such a chemically modified variant that can enhance the transferability of the antisense nucleotide into cells or the stability of the antisense nucleotide in the cells. Examples of such chemically modified variant are phosphorothioate, phosphorodithioate, alkyl phosphotriester, alkyl phosphonate, alkyl phosphoamidate and the like derivatives, and such variants may be prepared in accordance with known references (xe2x80x9cAntisense RNA and DNAxe2x80x9d, WILEY-LISS, 1992, pp. 1-50; J. Med. Chem., 36, 1923-1937 (1993)).
When introduced into cells, the above antisense nucleotides or chemically modified variants thereof can inhibit expression of a protein of the present invention. mRNAs produced by usual gene transcription are sense-strands, and the antisense nucleotides or chemically modified variants thereof can bind to such a sense-strand mRNA in cells to inhibit translation of the particular mRNA, resulting in inhibition of production of Semaphorin of the present invention. Because of such an effect of the antisense nucleotides or chemically modified variants thereof, they may serve as CNS-neuron regeneration promoters as described below.
It can easily be determined whether a particular antisense nucleotide prepared, or a chemically modified variant thereof, has a desired inhibitory effect or not, for example, by one of the following two methods. In one method, an antisense oligonucleotide or chemically modified variant thereof itself is directly introduced into cells expressing a novel Semaphorin of the present invention, and change in expression of said Semaphorin is then used as an indicator. In the other method, a gene capable of producing, when transcribed, said antisense RNA is introduced into the above Semaphorin-expressing cells, and change in expression of said Semaphorin is then used as an indicator.
Preferred examples of antisense nucleotides having such an expression-inhibiting effect are those oligonucleotides having sequences complementary to the transcription initiation site, translation initiation site, 5xe2x80x2 noncoding region, exon-intron junction region, or 5xe2x80x2 CAP region.
Antibodies of the present invention may be either polyclonal or monoclonal antibodies which specifically bind to a protein or peptide of the present invention. Such antibodies can easily be produced by immunizing an animal such as mouse or rabbit using a protein or peptide of the present invention in whole or in part according to the procedures described in, for example, xe2x80x9cCurrent Protocols in Immunologyxe2x80x9d, pp. 2.4.1-2.6.6 (J. E. Coligan ed., 1992). Fragments of antibodies obtained by purifying the above antibodies and degrading them with a peptidase are also included within the antibodies of the present invention. Such antibodies may be used, for example, in affinity chromatography or screening of cDNA libraries, and as pharmaceutical or diagnostic agents, or laboratory reagents.
The screening method for antagonists of a novel Semaphorin of the present invention refers to, for example, such a screening method of searching for substances which inhibits the neurite-outgrowth inhibition activity of a novel Semaphorin of the present invention. Such screening can be easily carried out by adding the above Semaphorin of the present invention to an assay system for Semaphorin activity described above, and further adding thereto a test substance (for example, a peptide, modified protein, low molecular weight compound or the like). In particular, inhibition of an activity (for example, the neurite-outgrowth inhibition activity) of Semaphorin of the present invention resulted from the addition of the test substance to the culture medium throughout the incubation period or only temporarily in the incubation period can be used as an indicator in the activity assay carried out with an added protein such as Semaphorin of the present invention. It is also important to confirm that the test substance alone does not influence the survival, neurite outgrowth and the like of neurons at the same concentration. When both of these requirements are fulfilled, one can consider the test substance as an antagonist of Semaphorin of the present invention. Although it is preferred to prepare in advance the test substance in the form of aqueous solution, an organic solvent such as DMSO may also be used as a solvent. In any cases, it is important to minimize the volume of solvent so as to exclude any effects of the solvent on neurons. Specifically, the volume to be added should be less than an equal volume, preferably less than {fraction (1/10)} volume, and more preferably less than {fraction (1/100)} volume relative to the culture medium. Some of antagonists of a novel Semaphorin of the present invention thus obtained will serve as CNS-neuron regeneration promoters as described below.
A transgenic animal of the present invention is produced by artificially inserting a gene or DNA of the present invention into the chromosome, or by knocking out said gene or DNA. Accordingly, the transgenic animals of the present invention include not only animals which expresses DNA of the present invention, but also so-called knockout animals in which DNA of the present invention is destroyed and dysfunctional. Destruction of DNA may be achieved by a known method (Shinichi Aizawa, xe2x80x9cGene Targetingxe2x80x9d, Yodosha, 1995).
Animals used herein may be those aminmals other than human, for example, laboratory animals such as mouse, rat, hamster, and rabbit, or domestic animals such as bovine and swine, and mouse is suitably used because of remarkable development of cell technology for mouse.
Methods for producing a transgenic animal are briefly described below for a transgenic mouse by way of illustration. For example, in the first method, DNA is microinjected into a pronucleus of an fertilized ovum of mouse. In the second method, DNA is introduced by infecting an eight cell stage embryo with a recombinant retrovirus. In the third method, DNA is introduced into an embryonic stem cell (ES cell) having totipotency, for example, by electroporation, and the cell is injected into another blastula to produce a chimera. See, xe2x80x9cManipulation of Mouse Embryoxe2x80x9d, B. Hogan et al. ed., 1986, Cold Spring Harbor Laboratory, and Shinichi Aizawa, xe2x80x9cGene Targetingxe2x80x9d, 1995, Yodosha.
Transgenic animals produced by the above methods are quite useful as an animal model for developing pharmaceutical agents or an animal used for a screening of pharmaceutical agents.
The usefulness of the novel Semaphorin and others of the present invention (proteins, DNAs, or peptides of the novel Semaphorin, antisense nucleotides or antibodies against the Semaphorin, and transgenic animals) is described below.
1) Usefulness as reagents in relevant area of investigation
While Semaphorin of the present invention inhibits neurite-outgrowth as described above, it has been also suggested that Semaphorin gene may have other unknown functions such as immunosuppression (Cell, 75, 1389-1399 (1993)). Accordingly, it is quite important for studies in relevant field to investigate expression of Semaphorin gene or distribution and function of Semaphorin protein. The present invention can provide DNAs, proteins, peptides, antibodies, antisense nucleotide, transgenic animals and the like which can be used for such purposes. Since the novel Semaphorin of the present invention is a secretory protein, it may be advantageously used as laboratory reagents.
2) Usefulness as pharmaceutical or diagnostic agents
One embodiment of the protein of the present invention is a protein which inhibits neurite outgrowth. Accordingly, such a protein may serve as therapeutic agent for immune diseases such as atopic dermatitis, pain or other diseases by virtue of their inhibition activity on neurite outgrowth of PNS-neurons.
In addition, as described in the xe2x80x9cBackground Artxe2x80x9d section, it has been recently suggested that a certain Semaphorin may play an important role also in peripheral non-nervous systems. In particular, it has been suggested that a certain Semaphorin may act in inhibiting the growth of cardiac muscles (Nature, 383, 525-528 (1996)). Also in the immune system, a certain Semaphorin has been suggested to be involved in survival and aggregation of B lymphocytes (Proc. Natl. Acad. Sci. USA, 93, 11780-11785 (1996)). It has also been suggested more recently that a certain Semaphorin may play some role in the immune reactions in rheumatism (B.B.R.C., 234, 153-156 (1997)). Involvement of Semaphorin in lung cancer has also been suggested (Proc. Natl. Acad. Sci. USA, 93, 4120-4125 (1996)).
Furthermore, it is believed because of the following reasons that Semaphorin of the present invention can inhibit cell movement such as cell migration or infiltration. While actin cytoskeleton plays an important role in movement of cell itself, it has been demonstrated that actin cytoskeleton similarly plays an important role also in movement of growth cone of neurons. It has been also demonstrated that formation of actin cytoskeleton in both cases is regulated by similar mechanisms in which G protein belonging to the Rho family is involved (Genes Develop. 8, 1787-1802 (1994); Cell, 81, 53-62 (1995)). Semaphorin of the present invention is presumed to inhibit elongation of growth cone via depolymerization of actin, and it is also conceivable that the same can inhibit movement (such as migration or infiltration) of cells expressing receptors for Semaphorin through similar mechanism. This argument is also supported by an observation that Ephrin known as a neuron guidance factor causing collapse of growth cone as Semaphorin did inhibit migration of neural crest cells (Neuron, 18383-396 (1997)).
In view of the above findings taken together, it is expected that the novel Semaphorin proteins, DNAs and the like of the present invention may inhibit infiltration or migration of cancer cells or immunocytes, and therefore, may be used as antiallergic agents, immunosuppressive agents, or anti-tumor agents.
In addition, as described below in Example 2, the novel Semaphorin of the present invention is expressed in a wide range of peripheral tissues and in a localized manner, suggesting that it may play some role at these sites or in the neighborhood thereof. In that case, the novel Semaphorin of the present invention may serve as therapeutic or diagnostic agent for diseases at the sites of expression indicated in Example 2.
As described in the above section 1), since the novel Semaphorin of the present invention is a secretory protein, it may be advantageously used as pharmaceutical or diagnostic agents.
On the other hand, it is suggested that a substance which inhibits the binding of natural Semaphorin to Semaphorin receptors may act as an antagonist inhibiting the neurite-outgrowth inhibition effect of Semaphorin. Since Semaphorin of the present invention is expressed also in the central nervous system, it is also presumed to be a CNS-neuron regeneration inhibitor. In that case, peptides, antibodies, or antisense nucleotides having an ability as an antagonist may promote regeneration of CNS-neurons, and therefore, may serve as therapeutic agents for spinal cord injury etc. Such antagonists include those substances found by the screening method described above.
The novel Semaphorin and other substances of the present invention, of which usefulness as pharmaceutical or diagnostic agents was described above, may be administered in doses and by administration methods as described below. For example, Semaphorin proteins, peptides, or antibodies may be formulated with an appropriate stabilizing agent, buffer and/or diluent, and used in an amount of several hundreds xcexcg to 2 g, and preferably of several tens mg or less, per administration. To reduce the administration frequency, it is possible to use a sustained release preparation, or to administer a formulation by portions over a prolonged period by means of, for example, an osmotic pump. Alternatively, cells expressing said Semaphorin protein or other substances may also be implanted into a living body for that purpose.
In the case in which an antisense nucleotide is used as pharmaceutical agents, such an antisense oligonucleotide or chemical variant thereof may be administered as such or an antisense RNA may be produced in cells, with doses and administration methods as described below.
In the method in which an antisense oligonucleotide or chemically modified variant thereof is administered as such, the antisense oligonucleotide preferably has a length of, for example, 5-200 bases, more preferably 8-25 bases, and especially preferably 12-25 bases. Antisense oligonucleotide or chemically modified variant thereof may be formulated by mixing it with a stabilizing agent, buffer, solvent and the like prior to its administration. Such formulation may optionally be co-administered with, for example, an antibiotic, anti-inflammatory, or anesthetic agent. Although the formulation thus prepared may be administered via various routes, it is preferred to topically administer at the site disordered. To avoid frequent administrations, a sustained release mini-pellet preparation may be prepared and embedded near the affected site. Alternatively, a formulation may be gradually and continuously administered to the affected site by means of, for example, an osmotic pump. The dose is typically adjusted so that the concentration at the site of action will be 0.1 nM to 10 xcexcM.
In the method in which an antisense RNA is produced in cells, the antisense RNA preferably has a length of, for example, more than 100 bases, preferably more than 300 bases, and more preferably 500 bases or more.
The methods by which a gene expressing an antisense RNA is introduced into a patient include an in vivo method in which the gene is directly introduced into cells in a living body, and an ex vivo method in which the gene is introduced into particular cells ex vivo and the cells are returned into the body (Nikkei Science, April, 1994, pp. 20-45; Gekkan-Yakuji, 36 (1), 23-48 (1994); Jikkenn-Igaku-Zokan, 12 (15), 1994; and references cited therein). An in vivo method is more preferred.
Such in vivo methods include a method employing recombinant viruses and other methods (Nikkei Science, April, 1994, pp. 20-45; Gekkan-Yakuji, 36 (1), 23-48 (1994); Jikken-Igaku-Zokan, 12 (15), in its entirety (1994); and references cited therein).
The methods employing recombinant viruses may include a method in which Semaphorin gene is incorporated into a virus genome of, for example, retrovirus, adenovirus, adeno-associated virus, herpesvirus, vaccinia virus, poliovirus, or sindbis virus, and the recombinant virus is introduced into a living body. Among these methods, those employing retrovirus, adenovirus or adeno-associated virus are particularly preferred.
Other methods may include a liposome method or a lipofectin method. The liposome method is particularly preferred.
For the ex vivo methods, microinjection, the calcium phosphate method, electroporation and the like may also be used, besides those techniques described above.
Administration of the gene to a patient is carried out via appropriate routes depending on, for example, the particular disease or symptom to be treated. For example, it may be administered intravenously, intraarterially, subcutaneously, or intramuscularly, or directly administered into the affected site such as nerve. For example, when spinal cord is infected with the recombinant virus, the expression of Semaphorin gene is inhibited exclusively in the spinal cord. Expression of an antisense nucleotide of the present invention typically lasts several days to several months, and such single infection is sufficient to allow regeneration of neuron. Re-infection may also be possible, when the antisense nucleotide is weakly expressed. When administered by an in vivo method, the gene may be formulated in the form of, for example, a solution, and typically it is formulated in the form of an injection containing an antisense nucleotide as an active ingredient to which conventional carrier or other additives may be added as needed. In the case of liposomes or membrane-fused liposomes (such as Sendai virus (HVJ)-liposomes) containing an antisense nucleotide, the liposome preparations may be in the form of a suspension, a frozen preparation, a centrifugally-concentrated frozen preparation or the like.
Although the amount of antisense nucleotide in the formulation may vary depending on, for example, the disease to be treated, the age and weight of the patient, it is typically 0.0001-100 mg, and preferably 0.001-10 mg. Such formulation is preferably administered once every several days to several months.